Neradova et al. BMC Nephrology (2017) 18:149 DOI 10.1186/s12882-017-0560-3

RESEARCH ARTICLE Open Access Phosphate binders affect vitamin K concentration by undesired binding, an in vitro study A. Neradova1* , S. P. Schumacher2, I. Hubeek3, P. Lux4, L. J. Schurgers4 and M. G. Vervloet1,5*

Abstract Background: Vascular calcification is a major contributing factor to mortality in end stage renal disease (ESRD). Despite the efficacy of phosphate binders to improve hyperphosphatemia, data on vascular calcification are less clear. There seems to be a difference in attenuation or delay in progression between different binders. In this in vitro experiment we tested whether phosphate binders could limit bioavailability of vitamin K2 by undesired binding. Vitamin K-deficiency limits activation of the vascular tissue mineralization inhibitor matrix γ- carboxyglutamate (Gla) protein (MGP) thereby exacerbating vascular calcification. Methods: In this experiment vitamin K2 (menaquinone-7; MK-7) binding was assessed by adding 1 mg of vitamin K2 to a medium with pH 6 containing 67 mg phosphate binder with either 7 mg of phosphate or no phosphate. Five different phosphate binders were tested. After five and a half hours vitamin K was analyzed by HPLC. All experiments were performed in triplicate. Results: Sucroferric-oxyhydroxide and sevelamer carbonate did not significantly bind vitamin K2, both in solution only containing vitamin K2 or in combination with phosphate. Calcium acetate/magnesium carbonate binds vitamin K2 strongly both in absence (p = 0.001) and presence of phosphate (p = 0.003). Lanthanum carbonate significantly binds vitamin K2 in solution containing only vitamin K2 (p = 0.005) whereas no significant binding of vitamin K2 was observed in the solution containing vitamin K2 and phosphate (p = 0.462). Calcium carbonate binds vitamin K2 significantly in a solution with vitamin K2 and phosphate (p = 0.009) whereas without phosphate no significant binding of vitamin K2 was observed (p = 0.123). Conclusions: Sucroferric-oxyhydroxide and sevelamer carbonate were the only binders of the five binders studied that did not bind vitamin K2 in vitro. The presence or absence of phosphate significantly interferes with vitamin K2 binding so phosphate binders could potentially limit bioavailability vitamin K2. Keywords: Phosphate binders, Phosphate, Vitamin K2 binding, In vitro

Background recent study supports the assumption that density, besides Chronic kidney disease (CKD) is a major and independent the volume of calcification is an independent risk factor risk factor for cardiovascular disease (CVD) [1]. During for dismal outcome [4]. Hyperphosphatemia is associated CKD disease progression, mortality risk due to CVD rises with VC [5]. Despite the widespread use of phosphate progressively and accounts for 50% of all deaths in the binders to control serumphosphate, evidence from CKD setting [2]. Vascular calcification (VC), almost prospective trials showing improved outcome by the use universally present in late stage CKD, is an important of phosphate binders is lacking. In fact it is unknown if contributor to CVD and CKD-related mortality [3]. A very the use of phosphate binders, especially calcium based binders, may actually be harmful [6]. There is some attenuation or delay of progression of VC by the non- * Correspondence: [email protected]; [email protected] 1Department of Nephrology, VU University Medical Center, De Boelelaan calcium-based phosphate binders as compared to the cal- 1117, 1081 HV Amsterdam, The Netherlands cium containing binders [7–9]. However VC frequently Full list of author information is available at the end of the article

© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Neradova et al. BMC Nephrology (2017) 18:149 Page 2 of 5

progresses despite adequate phosphate concentrations 5 min at 1800 rates per minutes and 20 degrees Celsius [10]. This has been attributed to increasing calcium load and after that 3 ml of supernatant was used for analysis. from calcium based binders [11], chronic inflammation [12] and other factors such as vitamin K deficiency [13]. Vitamin K measurement The prevalence of vitamin K deficiency is high in ESRD HPLC, C-18 reversed phase column and fluorometric patients [14]. Adequate levels of vitamin K are mandatory after post column electrochemical reduction was used for activation of matrix γ-carboxyglutamate (Gla) protein to determine the free vitamin K2 (MK-7) concentra- (MGP) [15]. MGP is secreted by chondrocytes and vascular tion. This method does not detect bound vitamin K2. smooth muscle cells in the arterial medial layer. MGP The inter-run variations as reported before were requires posttranslational modification by carboxylation between 6 and 8% [18]. and phosphorylation to become a functional calcification inhibitor of VC. We hypothesized that phosphate binders inhibit gastrointestinal uptake of vitamin K2 by undesired Materials binding since the binding of fat soluble vitamins has been Phosphate binders used are shown in Table 1. The abbre- described previously [16, 17]. If confirmed this could vations used are calcium carbonate (CC) lanthanum contribute to vitamin K deficiency and as such possibly to carbonatehydrate (LA) calcium acetate/magnesium car- the progress of vascular calcification. bonate (CA/MC), sucroferric-oxyhydroxide (SOH) and sevelamer carbonate (SC). Ammonium phosphate from Methods Sigma-Aldrich Co. (St. Louis, USA) was used for phos- Setup phate solutions. Ammonia 25% and hydrochloric acid 37% In an in vitro setup, vitamin K2 (synthetic menaquinone-7; were purchased from Merck (Darmstadt, Germany) and MK-7, kindly provided by Nattopharma ASA, Hovik, BDH (Fontenay-sous-Bois, France) respectively and Norway), was mixed with several contemporary available used to modulate pH of the medium. A Purelab Flex phosphate binders, in presence or absence of phos- water purification device of Elga Labwater Global phate, and incubated at pH 6 and fixed temperature Operations/Veolia Water Solutions & Technologies of 37 degrees Celsius. Vitamin K2 levels were mea- (United Kingdom) provided purified water for the sured after 330 min. All experiments were performed phosphate solutions and pH media. in triplicate. The tubes, wrapped in aluminum foil, contained a stirring bar and were placed in a glass Preparation of phosphate binders, phosphate solution, tank filled with water, which was placed on a magnet vitamin K2 (MK-7) and pH media stirrer, type RET-GS of IKAR Lab technik and the It is not well established what amount of different binders temperature was kept at 37° by an immersion circula- in in vitro setup represent comparable phosphate binding tor model 1112A of VWR International BV. The capacity in vivo. The amounts of phosphate binders and stirring bar allowed continuous stirring of the solu- phosphate solution were based on half the dosage, which tion and the heating kept the temperature at 37 has been used in previous in vitro experiments without degrees Celcius measured by an Easy-readR alcohol vitamin K2 [19]. Therefore, all substances were used in an based thermometer in a similar tube filled with a equal amount of active compound on weight base, and in stirring bar and the same amount of the same phos- powder form. CC, CA/MC and SOH tablets were crushed phate solution. After 330 min, 1 sample of 4 ml was with a pestle. We measured weight and used this value to taken out of each tube, initially containing 148 mcg calculate the amount of supplement needed to get 67 mg vitamin K2 (37 mcg/ml solution). Each sample was of the active compound, showed in formula 1. The put in a Rotanta 46R centrifuge of Hettich Zentrifugen for concentration of the active compound of the binders was

Table 1 Different phosphate binders, manufacturer and the city and country of origin Phosphate binder Manufacturer City, Country Lanthanum carbonatehydrate (FosrenolR 750 mg sachets) Shire Pharmaceutical Basingstoke, United Kingdom Calcium acetate/magnesium carbonate (OsvarenR 435 Fresenius Medical Care Bad Homburg, mg/235 mg film-coated tablets) Nephrologica Germany Sevelamer carbonate (RenvelaR 2,4 g sachets) Sanofi Europe B.V. Naarden, Netherlands Calcium carbonate (500 mg chewing tablets) Fagron BV Uitgeest, Netherlands sucroferric-oxyhydroxide (VelphoroR 500 mg chewing tablets) Vifor Fresenius Medical Neuilly-sur-Seine, Care; Renal Pharma France France Neradova et al. BMC Nephrology (2017) 18:149 Page 3 of 5

2.5 mg/ml. The needed amount of supplement was mea- Table 3 Vitamin K2 concentration is solution with vitamin K2, sured by a Mettler AT250 microbalance, with maximum phosphate and phosphate binders deviation of 0.05 mg. Vitamin K2 concentration P-value compared mcg/ml (SD) to control W ¼ ðÞÂA=P 67 control 27.53 (4.89) CC 0.497 (0.86) 0.009 Formula 1: W = used amount of substance (mg); LA 21.15 (11.94) 0.462 A = average weight (mg) of the whole supplement CA/MC 1.84 (3.19) 0.003 calculated after 10 measurements; P =amount of active SOH 41.8 (10.80) 0.135 component of substance (mg) mentioned on the package Two solutions were prepared pH 6 at start of the SC 32.9 (11.91) 0.528 experiment. One contained 25 ml of purified water and First column depicts the mean values of measured Vitamin K2 concentrations in mcg/ml in a solution containing vitamin K2, phosphate and a phosphate 1 ml of isopropanol in which 1000 mcg vitamin K2 was binder. P value is the comparison between control and phosphate binder. CC dissolved. Concentration of vitamin K2 was 37 mcg/ml. Calcium carbonate, LA Lanthanum carbonatehydrate, CA/MC calcium acetate/ magnesium carbonate, SOH sucroferric-oxyhydroxide and SC The second solution in addition contained 2.5 mM sevelamer carbonate phosphate to test potential competition of binding. The vitamin K2 content was also measured in solutions con- CC. Vitamin K2 concentrations were also measured in all taining only a phosphate binder or a phosphate binder solutions that did not contain vitamin K2 as a negative with phosphate to exclude interference of the matrix on control. In none of these samples vitamin K2 was HPLC with the measuring apparatus. detected. Solutions containing only phosphate with or without a binder only showed negligible vitamin K2 Data analysis concentration, excluding interference of phosphate or Statistical analysis was performed using SPSS 23. An phosphate binders with the vitamin K2 measurement independent sample T-test was used to compare the (data not shown). mean values of measured vitamin K2 concentrations. Discussion Results In this in vitro study we demonstrate that calcium- All phosphate binders, with the exception of SOH did containing phosphate binders and lanthanum carbonate bind vitamin K in the absence of phosphate. This decline bind vitamin K2. For LA this binding depends on the of free K2 concentration was statistically significant for absence of phosphate, pointing to competitive binding LA and CA/MC. In the presence of phosphate, K2 binding between phosphate and vitamin K2 for this compound. was attenuated for SC and LA, but increased for CC. For calcium carbonate this vitamin K2 binding was Table 2 depicts the vitamin K2 concentrations with only statistically significant in the presence of phosphate vitamin K2 and a phosphate binder in the solution. There compared to the solution without phosphate. In the is significant binding of vitamin K2 to LA and CA/MC. mixture with sevelamer carbonate a nominally lower Vitamin K2 concentrations in a solution containing concentration of K2 was shown as well, but this decline vitamin K2, phosphate and a binder are shown in Table 3. was not statistically significant. Addition of sucroferric- There is significant binding of vitamin K2 to CA/MC and oxyhydroxide did not lead to any decline of vitamin K2 at all, irrespective of presence or absence of phosphate. Table 2 Vitamin K2 concentration in solution with vitamin K2 Our study design precludes a formal quantitative com- and phosphate binders parison between the different phosphate binders. Several P Vitamin K2 concentration -value compared remarkable differences however appear obvious. First, mcg/ml (SD) to control both calcium containing compounds had increased affinity control 34.3 (4.35) for vitamin K2 in the presence of phosphate. We did not CC 25.9 (5.86) 0.123 study the chemical explanation for this feature, but LA <0.001a (<0.001) 0.005 possibly the formed calciumphosphate salt itself binds K2 CA/MC 6.47 (3.51) 0.001 as well. For lanthanum carbonate there appears to be SOH 47.27 (8.71) 0.106 competition for binding between phosphate and vitamin SC 19.79 (11.43) 0.147 K2. Although we could not find a statistically significant amount of vitamin K2 binding by sevelamer carbonate, First column depicts the mean values of measured Vitamin K2 concentrations in mcg/ml in a solution containing only vitamin K2 and a phosphate binder. where a previous study did [17], we did observe a trend in P value is the comparison between control and phosphate binder. CC the same direction. This may be due to difference in Calcium carbonate, LA Lanthanum carbonatehydrate, CA/MC calcium acetate/ magnesium carbonate, SOH sucroferric-oxyhydroxide and SC sevelamer carbonate. a concentrations used: 2.5 mg/ml and 4 mg/ml for sevelamer this value represents a figure below the detection limit, which was 40 pg/ml cabonate; and 37 mcg/ml (our study) and 0.5 and 5 ug/ml Neradova et al. BMC Nephrology (2017) 18:149 Page 4 of 5

(Takagi et al.) for vitamin K. Moreover the previous study we mainly aimed to test the proof of principle of the research did not report whether vitamin K1 or K2 was used existence of vitamin K2 binding by contemporary phos- as substrate [17]. SOH showed no vitamin K2 binding at phate binders. Obviously, the clinical relevance of our all. The statistically non-significant higher values of vitamin findings cannot be assessed in an in vitro setup. Another K2 when using the latter binder, as compared to control is limitation, as described, is the fact that no quantitative probably the consequence of assay variability. Extrapolating comparison between phosphate binders can be made. these results to biological systems, including the treatment of hyperphosphatemia in CKD patients can only be done Conclusion with great caution. However, our data do point to a feature Sucroferric-oxyhydroxide and sevelamer carbonate were of some commonly used phosphate binders that may have the only binders of the five binders studied that did not clinical consequences. Vitamin K deficiency results in bind vitamin K2 in vitro. The clinical significance of this undercarboxylation of MGP, and as such limit natural finding requires additional studies. defense against ectopic calcification including VC. To some extent, the enigmatic lack of improvement of relevant Abbreviations endpoint in clinical studies, despite effective phosphate CA/MC: Calcium acetate/magnesium carbonate; CC: Calcium carbonate; lowering potential, could be attributed to a decline in CKD: Chronic kidney disease; CVD: Cardiovascular disease; LA: Lanthanum bioavailability of vitamin K from the gastrointestinal tract carbonatehydrate; MK-7: Synthetic menaquinone-7; SC: Sevelamer carbonate; SOH: Sucroferric-oxyhydroxide; VC: Vascular calcification by phosphate binder therapy. In recent years some studies suggested superior clinical outcome when using sevelamer Funding carbonate as compared to calcium containing binders Research was supported by a Grant from the Dutch Kidney Foundation. [20, 21]. Generally this has been ascribed to the calcium There was no role of the funding body in collection, analysis, interpretation loading as a consequence of calcium content in these of data or in the writing of the manuscript. binders. Our data suggest an additional mechanistic Availability of data and materials explanation, which is the increased likelihood of The datasets analysed during the current study are available from the vitamin K deficiency. In support of this hypothesis is corresponding author on reasonable request. also a study in which non-hyperphosphatemic CKD patients were treated with either placebo or phosphate Authors’ contributions binder therapy. Despite a small decline in serum phos- AN wrote the manuscript and designed the laboratory set. SS worked in the laboratory, performed the experiment and revised the manuscript. IH helped phate there was an increase in arterial calcification in with the laboratory work as supervisor and revised the manuscript. PL the active treatment arm [10]. Our finding that vitamin analyzed the vitamin K2 content with the HPLC. LS was a major contributor K2 binding by LA is absent in presence of phosphate to writing the manuscript and helped with designing the laboratory set up. MV was a major contributor in writing the manuscript and helped with the does not necessarily imply that in patients no relevant laboratory set up. All authors read and approved the final manuscript. vitamin K binding occurs. It is likely that high gastro- intestinal levels of phosphate do not always match local Competing interests high concentrations of phosphate binders. In addition, A. Neradova was awarded a grant from the Dutch Kidney Foundation. M. dose up titration in case of persistent hyperphosphatemia Vervloet received consulting fees from Vifor-FMC Renal pharma; Otsuka, Amgen, lecture fees from Amgen and Baxter and has obtained grants from may increase likelihood of unnoticed aspecific binding of Amgen, Dutch Kidney Foundation, Shire, Sanofi and Pfitzer. Other authors non-phosphorus compounds. This may especially apply in have no competing interests to declare. hyperparathyroidism where a substantial amount of phos- phate may be bone-derived. Our study has some limita- Consent for publication tions. We used a fixed pH which does not necessarily Not applicable. match the gastrointestinal tract, where pH may be as low Ethics approval and consent to participate as 1 in the stomach. However, at sites where vitamin K2 Not applicable. uptake occurs pH is not low, so our set-up may be more relevant than at low pH. A single measurement of vitamin ’ K2 was performed at 330 min which was considered a Publisher sNote Springer Nature remains neutral with regard to jurisdictional claims in reasonable period to mimic the time needed for the published maps and institutional affiliations. passage to the terminal ileum where the absorption of vitamin K2 starts [22]. Furthermore, only vitamin K2 has Author details 1Department of Nephrology, VU University Medical Center, De Boelelaan been used in our experiments, since vitamin K2 is the 1117, 1081 HV Amsterdam, The Netherlands. 2Department of cardiology, VU preferred cofactor for the carboxylation of MGP [23]. In University Medical Center, Amsterdam, The Netherlands. 3Clinical chemistry addition, phosphate and vitamin K concentrations in vivo laboratory, VU University Medical Center Amsterdam, Amsterdam, The Netherlands. 4Department of Biochemistry, Cardiovascular Research institute may differ substantially, and moreover a wide range of Maastricht, University Maastricht, Maastricht, The Netherlands. 5Institute for additional aspects may be very different as well. In our Cardiovascular Research VU, ICaR-VU, Amsterdam, The Netherlands. Neradova et al. BMC Nephrology (2017) 18:149 Page 5 of 5

Received: 26 July 2016 Accepted: 19 April 2017

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